PROJECT SUMMARY Acute respiratory illnesses (ARI) are the leading cause of death in children under five years, with viral pathogens playing a major role, including adenovirus (AdV). Over 85 AdV types (adenotypes) have been described based on their unique genetic makeup and selected adenotypes are associated with ARI. In multiple respiratory surveillance studies in the United States (U.S.) and globally, AdV is frequently detected, including in asymptomatic individuals, sometimes at frequencies similar to symptomatic patients. Thus, it is unclear whether certain adenotypes are more prevalent in healthy asymptomatic individuals as compared to ARI patients. AdV causes a broad range of clinical syndromes in both normal and immunocompromised hosts, including pneumonia complicated by respiratory failure and even death. It is also unknown if certain adenotypes or a specific signature of virulence in certain adenotypes are associated with severe disease outcomes. Additionally, accurate data are needed on pediatric AdV disease burden across a wide geographic area and multiple seasons. Using the CDC-sponsored New Vaccine Surveillance Network (NVSN), a multi- center collaboration with geographical variation while retaining protocol consistency, we plan define epidemiology of AdV infection and circulation patterns of adenotypes in U.S. children over four respiratory seasons. The central hypothesis of this proposal is that there are multiple co-circulation of adenotypes in geographically diverse locales, and that specific adenotypes are associated with severe disease outcomes. The proposed research will define the burden and epidemiology of AdV-associated ARI in children over a broad geographic range and ascertain the relationship of AdV genetic diversity to disease-associated and asymptomatic infection through the following aims: SPECIFIC AIM 1: To determine spatiotemporal circulation patterns and prevalence of adenotypes in U.S. children with ARI compared to healthy controls. SPECIFIC AIM 2: To determine if adenotypes or sequence signatures of adenotypes are associated with disease severity. All positive AdV specimens from both ARI cases and healthy controls from the six other NVSN sites collected over four consecutive respiratory seasons will be sent to Vanderbilt. Real- time quantitative type-specific PCR assays for adenotypes 1-7, 11, 14, 16, and 21 will be performed. The frequency of these adenotypes, geographic location, and seasonal patterns will be compared between cases and controls. We will also determine if there are distinct adenotypes that are associated with severe illness. In addition, we will perform metagenomic sequencing on select (>200) specimens to obtain complete genome sequence of AdVs and perform phylodynamic analysis to identify specific adenotypes or sequence signatures associated with disease severity. Data from this study will fill important gaps related to the burden of various adenotypes associated with ARI and risk factors for severe disease.